The role of dietary soybean proteins in the control of lipidemic levels of hypercholesterolemic patients is a widely accepted issue [1]. In previous studies [2-4], the direct involvement of one subunit of the soybean 7S globulin, the α′ subunit, in the up-regulation of the LDL-receptor was demonstrated in in vitro and in vivo systems, suggesting that biologically active (poly)peptides, capable of modulating cholesterol homeostasis, are likely to be produced by cell enzyme processing.
The native 7S globulin is composed of three randomly assorted polypeptide chains, the α′, α and β subunits [5], encoded by different genes. The mature α′ (Accession N. P11827 UniProtKB/Swiss-Prot database) and α chains (Accession N. P13916 UniProtKB/Swiss-Prot database) share an extended N-terminal region of about 145 amino acid residues which is missing in the β subunit (Accession N. P25974 UniProtKB/Swiss-Prot database). Based on the peculiar amino acid sequence of the α′ extension region, this subunit was purified by metal affinity chromatography and orally administered to hypercholesterolemic rats, thus allowing to show both its plasma lipid-lowering properties and up-regulation of liver β-VLDL receptors [4]. On the other hand, since the molecular weight of the α′ subunit is around 71 kDa [6], it seems unlikely that it may cross in vivo the intestinal barrier with no modification.
For this reason, our research has been directed to seeking the amino acid sequence/s of α′ subunit responsible for the pharmacological effect. Since the core regions of the three subunits have more similar amino acid sequences, it is conceivable that the biological activity should reside in one or more (poly)peptides of the extension region. In principle, the localized but significant amino acid differences between the extension regions of the α′ and α chains would limit the number of peptides responsible for the biological activity.
From these previous statements, the first strategy pursued was to test the effect of both polypeptides in Hep G2 cells, obtained from the in vitro digestion (pepsin/trypsin) of CroksoyR70, an isoflavone-free soybean concentrate routinely employed in the dietary treatment of hypercholesterolemic patients [7-8], and synthetic peptides, corresponding to specific amino acid sequences that differed between the 7S soybean globulin subunits, on the LDL-receptor (LDL-R) modulation. The results obtained in these studies pointed out that a marked LDL-R up-regulation could be induced in HepG2 cells exposed to enzyme digestion products of CroksoyR70 with MW ranging from 3,000 to 20,000 Da, as well as to a small synthetic peptide (2,271 Da) from 7S soybean globulin added to cells at a concentration of 10−4M [9]. The study obtained with small peptides is still currently under investigation and have not been conclusive so far [10].
The cholesterol and triglyceride lowering capacity of soybean proteins is a consolidated issue. The soybean protein diet is currently the most potent dietary tool for treating hypercholesterolemic patients, thus providing a unique opportunity for the management of adults and very young subjects. Moreover, it is clearly established that the plasma cholesterol reduction is greater in patients having a high baseline degree of cholesterolemia [14].
The hypothesis that proteins per se reduce blood cholesterol arose from experimental studies indicating that a shift from animal to plant proteins in the diet activates the LDL receptor system in the liver of laboratory animals [15], as well as in circulating lymphomonocytes of hypercholesterolemic patients [16]. To identify the soybean protein components responsible for the cholesterol lowering effect, in vitro studies were carried out with a human hepatoma cell line that is highly sensitive to factors regulating LDL-receptor expression and cholesterol biosynthesis/breakdown. The purified α′ subunit from the 7S soybean globulin was found to up-regulate LDL-receptors in Hep G2 cells [3] and this finding was confirmed in cholesterol-fed rats [4]. Although these data support the hypothesis that the protein moiety is responsible for the observed biological effect, arguments may be raised to α′ chain in vivo biological fate, since peptides and amino acids are normally produced by the action of gastric and/or intestinal proteolytic enzymes. However, an increasing number of animal and plant (poly)peptides is being claimed to play relevant regulatory functions, often attributed to anti-oxidant, anti-proliferative and anti-inflammatory effects [17]. As far as soybean is concerned, experimental evidence clearly indicates the possibility that peptides and even small compact proteins, such as the Bowman-Birk inhibitor, may be adsorbed [18], thus eliciting a number of effects, including anti-cancer, anti-inflammatory, radio-protective ones [19]. Also genetically modified soybean (poly)peptides have been shown to trigger biological responses, such as hypotensive effects [20]. Recently, a LDL-R transcription stimulating peptide (FVVNATSN) (SEQ ID NO: 3), deriving from the 7S globulin β chain, has been identified from a soybean hydrolysate prepared by a protease from Bacillus amyloliquefaciens and then by chemical synthesis [21]. In this case, an increased LDL-R transcription (+148%) was detected in Hep G2 cells exposed to the peptide at a concentration of 100 μM. Other peptides arising from the 11 S globulin have been shown to exert similar but lower activity [21].
It would be desirable to make available shorter polypeptides maintaining or even improving the biological properties of the full length protein.